The control of protein folding, assembly, and transport from the endoplasmic reticulum (ER) is essential to the fidelity of cell surface events that regulate lymphocyte function and differentiation. In recent years, several resident ER proteins have been identified that appear to either aid or monitor protein folding and assembly, although the precise roles of these "molecular chaperones" in protein maturation is unclear. It is the central hypothesis of this project that the various ER chaperones play distinct and essential roles in the maturation of nascent Ig molecules and thus in B cell development and differentiation. The failure of Ig molecules to interact with components of the ER chaperon machinery could allow the secretion or surface deposition of aberrant Ig molecules, thus circumventing this controlled process. Most recently, it has been discovered that the amyloid deposits found in immunoglobulin deposition disease are caused by mutant Ig proteins. It is very possible that the mutations in these Ig proteins interfere with their interactions with ER chaperones and allow them to by-pass the control mechanisms for removing defective molecules from the secretory pathway. Experiments in Specific Aim 1 are designed to provide a clearer understanding of the molecular sequence of events in the folding and assembly of Ig heavy and light chains. In Specific Aim 2, the role of various ER chaperones in controlling the maturation and transport of Ig proteins will be elucidated. To achieve these aims, a battery of BiP ATPase mutants that bind Ig proteins in vivo but that cannot release them and thus act as "chaperon traps" have been generated and fully characterized. Both transient and inducible systems for the in vivo expression of these BiP mutants have been developed and cell lines that are deficient in the synthesis of various ER chaperones have been produced. In addition, cDNA clones for Ig heavy and light chains have been obtained and used to identify the in vivo folding intermediates for Ig light chains. Antisera specific for various ER chaperones were produced and cDNA clones for grp94 and ERp72 were obtained, which will permit reconstitution of the chaperon-deficient cell lines. Together, these reagents will allow delineation of the intermediates of the Ig folding and assembly pathways, identification of those proteins that directly aid these processes as well as those that monitor the reactions, and finally determination of the mechanisms for retention and degradation of unsuccessful Ig molecules. This information could be crucial to understanding immune pathologies such as light chain deposition disease.